Magnesium hexacyanoferrate nanocatalysts attenuate chemodrug-induced cardiotoxicity through an anti-apoptosis mechanism driven by modulation of ferrous iron

Distressing and lethal cardiotoxicity is one of the major severe side effects of using anthracycline drugs such as doxorubicin for cancer chemotherapy. The currently available strategy to counteract these side effects relies on the administration of cardioprotective agents such as Dexrazoxane, which unfortunately has unsatisfactory efficacy and produces secondary myelosuppression. In the present work, aiming to target the characteristic ferrous iron overload in the doxorubicin-contaminated cardiac microenvironment, a biocompatible nanomedicine prepared by the polyvinylpyrrolidone-directed assembly of magnesium hexacyanoferrate nanocatalysts is designed and constructed for highly efficient intracellular ferrous ion capture and antioxidation. The synthesized magnesium hexacyanoferrate nanocatalysts display prominent superoxide radical dismutation and catalytic H2O2 decomposition activities to eliminate cytotoxic radical species. Excellent in vitro and in vivo cardioprotection from these magnesium hexacyanoferrate nanocatalysts are demonstrated, and the underlying intracellular ferrous ion traffic regulation mechanism has been explored in detail. The marked cardioprotective effect and biocompatibility render these magnesium hexacyanoferrate nanocatalysts to be highly promising and clinically transformable cardioprotective agents that can be employed during cancer treatment.


REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): In this paper, MgHCF nanocatalysts (NCs) have been synthesized through a PVP-directed selfassembly method. The NCs possesses potent ferrous capturing and antioxidation functions for cardioprotection during tumor chemotherapy. The viability of the cardiomyocytes impaired by DOX could be effectively protected by the MgHCF NCs. The MgHCF NCs also exhibit excellent in vivo cardioprotection effect via attenuating the cardiotoxicity and the harmful side-effect due to the DOX contamination without deteriorating the anti-tumor effect of the chemodrug. The MgHCF NCs is novel and effective for the cardioprotection application. Their in vivo and in vitro effects have been thoroughly demonstrated. The paper is well organized. But the characterizations of MgHCF NCs are insufficient. This paper can be published after the following revisions: (1) In Scheme 1 and Figure S1, what do the green spheres and light blue thorny particles mean? They should be indicated in the figures.
(2) The detailed structure of MgHCF NCs is not clearly clarified. Actually, the schematic structure in Figure 1a is not supported by the data. More evidence should be supplied to clarify the structure of MgHCF NCs.
(3) In Figure 1d and 1e, the XRD patterns and FTIR spectra of pure PVP and MgHCF NCs should be supplied for better comparison.
(4) The ferrous capturing ability of MgHCF NCs should be proved under the interference of intracellular biomacromolecules. Thus the Mg2+ and Fe2+ exchange experiments should be performed in the presence of cell lysis to mimic the in vivo environments. (5) After ferrous capturing, how do the antioxidation performances of MgHCF NCs change? (6) In Figure S14, why do DCFH-DA-stained cardiomyocytes treated with DOX supplemented with MgHCF at the concentration of 80 ppm show much stronger fluorescence than that treated at the concentration of 40 ppm? And in Figure S15, DCFH-DA-stained cardiomyocytes treated with DOX supplemented with DXZ at the concentration of 25 ppm still showed much stronger green fluorescence, it cannot say that the oxidative stress is significantly relieved. (7) Beside the FerroOrange staining method, could the authors supply more quantitative data to measure the intracellular ferrous concentration?
Reviewer #2 (Remarks to the Author): Hou et al investigated the Magnesium Hexacyanoferrate Nanocatalyst attenuates chemodrug-Induced cardiotoxicity through ferromodulation-driven anti-apoptosis. Excellent in vitro and in vivo cardioprotection performances of MgHCF NCs have been demonstrated and the underlying intracellular ferrous traffic regulation mechanism has been explored in detail. The marked cardioprotective effect and biocompatibility render MgHCF NCs to be a highly promising and clinically transformable cardioprotective agent to be employed during cancer treatments. This is an interesting finding in the Dox induced cardiotoxicity treatment. However there are some questions need to be explained as follow: Major: 1. The stuides on the biological properties of MgHCF NCs is not sufficient, including the half-life of the drug in the blood, drug utilization, median lethal dose, and drug distribution in different organs after intraperitoneal injection of MgHCF NCs, especially the drug concentration in the heart and tumor should provided. 2. Long-term side effects of MgHCF NCs on the number of red blood cells and hemoglobin should be investigated? 3. The important question in this study is, why the authors didn't study cardiomyocyte ferropotosis, but apoptosis? There have been many reports about DOX induced myocardial ferropotosis. If MgHCF NCs can inhibit iron ions, the main target should be ferroptosis. 4. The H9c2 cells used in this study are derived from myoblast cells, which are closer to those of skeletal muscle cells. H9c2 cells have the ability to proliferate, which is significantly different from cardiomyocytes. So it is recommended that the author repeat the relevant experiments with primary cardiomyocytes. Minor: 1. The quality of Fig 4J Western blot is too poor. 2. Fig 6J LVEF% should not use normalized data, but should use raw data. 3. The caspase-3 in Figure is incorrectly labeled, it should be cleaved caspase-3. 4. In the in vivo data, n=4, which does not meet the statistical requirements. 5. The animal survival curve is that n=6-5 does not meet the statistical requirements. Generally, the number of animals in each group should be 10-20. 6. The detection data on autophagy, mitophagy, apoptosis and ferroptosis of cells and animals are insufficient. In short, this study is interesting, but not sufficient to be published in NC. More functional studies and pharmacological studies data should provided to support the Reviewer #3 (Remarks to the Author):

Key results
In cancer patients, treatment with anthracyclines (i.e., doxorubicin) is very often jeopardized by druginduced cardiotoxicities mostly caused by excessive cellular oxidative stress. Here, the authors clearly demonstrate that the accumulation of radical species is catalyzed by Fenton-like reactions consecutive to alterations in iron transport and report on the development of a new nanomedicine, called MgHCF NCs, specifically designed to capture iron and hence reduce the iron-associated risk. After demonstrating its efficacy in eliminating the cytotoxic radical species in vitro, the authors showed the biocompatibility and cardioprotection of MgHCF NCs in vitro (H9C2 cardiomyoblasts) and in vivo (mice). Finally, in a tumor mouse model, the authors demonstrate that MgHCF NCs do not impair the anticancer efficacy of doxorubicin while significantly reducing its cardiac side effects. Based on convincing data, they conclude that MgHCF NCs displays all properties for a promising cardioprotective agent during cancer treatments.

Validity
The general framework of the research is based on a proven clinical reality according to which patients exposed to anthracyclines, such as doxorubicin, develop cardiotoxic damage during treatment, compromising the chances of success. This adverse effect is caused by the generation of reactive oxygen species, creating oxidative stress induced by the anticancer agent. The phenomenon would be amplified by an alteration of iron transport in heart cells, creating an environment conducive to Fenton reactions catalyzing the genesis of free radicals. This hypothesis has been partially confirmed in previous studies showing the partially protective effect of an iron chelator, dexrazoxane, the only cardioprotective agent used clinically to date in this type of chemotherapy. In this publication, the authors present convincing results obtained in various in vitro and in vivo models showing the superior efficacy to dexrazoxane of a new nanomaterial, MgHCF NCs. The data clearly show that this nanocatalyst induces excellent cardioprotection thanks to its iron capture (by replacement of initially trapped magnesium ions) and antioxidant properties. In vitro, according to their transcriptomic and proteomic data acquired in the H9C2 line of cardiomyoblasts, the authors show that this nanocatalyst significantly increases the chances of survival of cardiac cells exposed to doxorubicin by reducing both deregulation of iron trafficking and cell apoptosis. In vivo (mice), this cardioprotective effect was partially confirmed by cardiac echocardiography studies. Finally, the authors presented in vivo data in mice showing the verified biocompatibility of the new agent, during repeated exposures over several weeks and also that the latter does not interfere with the anticancer capacities of doxorubicin in a mouse model implanted with a subcutaneous tumor. The encouraging results obtained for the new nanomaterial in terms of cardioprotection are supported by the fact that in the majority of in vitro and in vivo tests evaluated in this study, the latter performed better than dexrazoxane, used here as a positive benchmark. Overall, the authors' conclusions (claiming that their new MgHCF Ncs nanomaterial is one of the most promising cardioprotective agents for clinical use) are appropriately supported by the data, well justified and reliable. Significance To the best of my knowledge, the authors' working hypotheses on the cellular mechanism leading to doxorubicin-induced cardiotoxicity, as well as their conclusions regarding the efficacy of their new nanocatalyst as a promising cardioprotective agent during cancer therapy are correct. I am not aware of any publication supporting contradictory data.
Data and methodology First, I would like to mention that I do not have the expertise concerning the physicochemical aspects of characterization of the nanocatalyst at the center of this publication. I will therefore not comment on this part of the results. On the other hand, I feel comfortable with the evaluation of in vitro and in vivo tests of biocompatibility and efficacy of the product. Here are my main comments on this part : 1. Concerning the in vitro evaluation of MgHCF NCs antioxidant properties, I am not convinced with the use of "multi-enzymatic catalytic performance" (see line 196) as well as with the "SOD superoxide dismutase-and catalase-like catalytic activities" (see for example lines 30) of MgHCF NCs. Such wording should be avoided since the agent can indeed induce those effects but lacks enzymatic activity. 2. In the evaluation of MgHCF NCS' cardioprotective properties, the authors used both in vitro and in vivo assays. In the in vitro cellular experiments, they selected the H9C2 cell line and inappropriately called them "cardiomyocytes" (line 739). In fact, H9C2 cells are cardiomyoblasts which exhibit most of the phenotypic characteristics of mature heart cells except the contractile properties. As a result, they are less dependent on oxidative phosphorylation and mitochondrial activity, the targets of doxorubicin studied here. It would be interesting to reproduce these experiments in human cardiomyocytes, for example the AC12 cell line. 3. Now, regarding the in vivo data in mice, the biocompatibility was only assessed in a limited number of animals (n= 4/group), which seems too little for conduct appropriate statistical test. In addition, data on the MgHCF NCs' ADME properties are lacking to properly evaluate potential adverse effects. How fast the agent is absorbed after i.p. injection. What are the plasma halftime and Cmax. To what tissues/organs is the agent distributed ? What is the proportion of the injected compound reaching the target organ (heart) and what is the mechanism of uptake by cardiac cells. For a better risk assessment of the exposure to MgHCF NCs, it is also mandatory to know the expected efficacy dose. This could help in the determination of a margin of safety. Finally, the rationale behind the selection of doses (both for MgHCF NCs and dexrazoxane) used in the in vivo experiments should clearly be stated. 4. In the safety assessment of MgHCF NCs in vivo (mice), I regret that the authors did not investigate the potential effect of the magnesium released from nanomedicine during iron uptake. Indeed, a magnesium overload is known to cause cardiovascular effects, including cardiac cytotoxicity and hypotension. In most of the in vitro and in vivo assays (except maybe for the biocompatibility) presented in the study, the number of replicates and statistical tests are appropriate.
Analytical approach As already mentioned, the analytical methods used in the physico-chemical characterization of the nanomedicine are outside the scope of my expertise. Regarding other analytical approaches, I only have one specific comment related to the assessment of cardioprotection using echocardiography. I do believe that those registered cardiac parameters are not sufficient for a comprehensive risk assessment. In particular, due to the release of Mg++ ions in the cardiac cells during iron uptake by the nanocatalyst, I would recommend to add ECG-like recordings.

Suggested improvements
As already mentioned, additional information should be provided regarding : 1. Rationale for the selection of the doses (MgHCF NCs and dexrazoxane) used for the in vivo experiments 2. Better characterization of MgHCH NCs' ADME properties 3. Exposure of mature human cardiomyocytes to MgHCF Ncs (AC12 cell line) 4. Determine potential release of Mg2+ from nanocatalyst in blood compartment or in other tissues 5. Discuss the potential risk of Mg++ release in cardiomyocytes during iron uploading in the nanocarrier Minor changes : 1. Lines 41, 48 428 : replace "systematic" by "systemic" 2. Line 75 : replace "suffer from. The" by "suffer from the " 3. Line 81 : the sentence "remains limited chelation strengthen and capability" is not clear, please rephrase 4. Line 112 : replace "administration for during the" by "administration during the" 5. Line 195 : replace "and the simultaneous the magnesium " by "and simultaneously the magnesium " 6. Line 352 : replace "we next compare " by "we next compared" 7. Line 397 : replace "ameliorated MgHCF NCs " by "ameliorated by MgHCF NCs" 8. Line 620 : reword "has been being" 9. Line 739: replace "cardiomyocytes" by "cardiomyoblasts"

Clarity and context
The text is precise and perfectly understandable. The legends of the figures are self-sufficient to understand the graphics without necessary return to the text.

References
The references are appropriate and satisfactorily justify the points to which they are linked in the text.
Reviewer's expertise As already mentioned, I do not have the expertise concerning the physicochemical aspects of characterization of the nanomaterial developed and evaluated in this study.
In this paper, MgHCF nanocatalysts (NCs) have been synthesized through a PVP- (1) In Scheme 1 and Figure S1, what do the green spheres and light blue thorny particles mean? They should be indicated in the figures. (2) The detailed structure of MgHCF NCs is not clearly clarified. Actually, the schematic structure in Figure 1a is not supported by the data. More evidence should be supplied to clarify the structure of MgHCF NCs.
Response: Thank you for your comment. To better clarify the structure of MgHCF NCs, we have supplemented additional characterizations (XRD patterns and FTIR spectra) of MgHCF NCs and other material counterparts during the revision. From the supplemented XRD pattern of PVP, it is clear that the MgHCF NCs displays a similar pattern as PVP polymer ( Figure S4). From the supplemented FTIR spectra, we can find that the spectrum of MgHCF NCs contains all peaks belonging to PVP polymer with additional C≡N stretching vibrations (updated Figure 1e). Based on the interactions of PVP-Mg 2+ as well as PVP-ferricyanide ions, we therefore confirm that the MgHCF NCs are amorphous nanoparticles assembled from the ferricyanide and magnesium ions through the interaction with polyvinyl pyrrolidone polymer. Additionally, we have also revised the graphic of MgHCF NCs for better clarification throughout the manuscript.
(3) In Figure 1d and 1e, the XRD patterns and FTIR spectra of pure PVP and MgHCF NCs should be supplied for better comparison.  Response: Thank you for your comment and suggestion. To better imitate the intracellular environment containing abundant biomacromolecules, the authors have employed RPMI full media with 10% fetal bovine to initiate the cationic exchange experiment. From the ICP results of the magnesium and iron concentrations, we can find that the ferrous capturing ability of MgHCF NCs will not be affected by the biomacromolecules interference. Data are presented in Figure S5 in the revised Supporting Information.
(5) After ferrous capturing, how do the antioxidation performances of MgHCF NCs change?
Response: Thank you for your suggestion. To address your concern, we initially synthesized MgHCF NCs followed by the ferrous addition to form PB NPs. We then evaluated the antioxidation performance of the formulated PB NPs by using a dissolved oxygen meter as well as a SOD-assay kit. Supplemented data has been presented in We also agree with the reviewers that pharmacokinetic performance of MgHCF NCs is the most pivotal evaluation for their clinical translation. Therefore, we have supplemented the half-terminal time ( Figure S41a) and tissue biodistribution experiments of MgHCF NCs (Figure S41b). We found that upon MgHCF NCs administration, MgHCF NCs are mainly distributed into liver, spleen and lung in 2 h post-injection. In 12 h and 24 h post-injection, the overall distribution amounts of Mg 2+ into these major organs were gradually decreased. Specifically, the distributed percentages of Mg 2+ into heart were determined to be 7.16 ± 0.39 ID %/g, 5.39 ± 1.77 ID %/g and 1.95 ± 0.27 ID %/g respectively. For tumor accumulation, 2.84 ± 0.84 ID %/g, 1.23 ± 0.45 ID %/g and 1.20 ± 0.52 ID %/g could be determined in 2 h, 12 h and 24 h post-injection, respectively. Relevant discussion has been supplemented in the revised Manuscript (Page 25).
We where w represents the drug utilization; C0 represents the plasma concentration of Mg 2+ at t = 0; ID represents the injection dose, i.e., 4.8 mg kg -1 ; m represents the weight of mice; V represents the total circulation blood volume of a mice.

Long-term side effects of MgHCF NCs on the number of red blood cells and hemoglobin should be investigated?
Response: Thank you for your suggestion. We have supplemented the blood routine assays for mice in one-month post administration with multiple doses of MgHCF NCs.
The results have been supplemented as Figure S31 in the revised Supporting Information. From the blood routine assays, unaffected red blood cells and hemoglobin levels could be determined, revealing the satisfactory long-term biosafety of MgHCF NCs.
3. The important question in this study is, why the authors didn't study cardiomyocyte ferroptosis, but apoptosis? There have been many reports about DOX induced myocardial ferroptosis. If MgHCF NCs can inhibit iron ions, the main target should be ferroptosis.
Response: Thank you for your comment. According to the previous literatures, several programmed cell death pathways such as apoptosis and ferroptosis would participate in the pathology of doxorubicin-induced cardiotoxicity. We have investigated the cell apoptosis origin by evaluating intracellular oxidative stress, as well as the pro-apoptosis regulations in mRNA and protein aspects. In addition, intracellular abnormal iron accumulations and ferroptosis-associated biomarkers have been studied. As the intracellular iron regulations play an important role in both cell apoptosis and ferroptosis, we have performed detailed investigations on both apoptosis and ferroptosis.
From our cellular mRNA sequencing results, we can observe combined cell death pathways for DOX-contaminated cardiomyocytes such as apoptosis, ferroptosis, autophagy and mitophagy, with apoptosis being the most dominant induced by DOX chemodrug. Although the transcriptome regulations by MgHCF contribute to the enrichment of ferroptosis KEGG pathway with significance (Figure 4), the scored gene ratio (0.779 %) remained relatively low (compared to 2.336 % of apoptosis). According to the highly up-regulated GPX4 which encodes glutathione peroxidase for lipid peroxidation clearance in DOX pathology, the pathological regulation of intracellular iron is believed to be less significant to cause prevailing cell ferroptosis judged from the non-destruction of anti-lipid peroxidation system. Based on the above considerations, we believed that apoptosis is still the main cell death pathway during DOX-induced pathology. and their corresponding references have been reconducted. Higher quality of the bands could be observed in the updated Figure 4J in the revised Manuscript. Protein quantification has also been updated ( Figure S27). Figure 4J has been improved and updated.

The resolution of
2. Fig 6J LVEF% should not use normalized data, but should use raw data. 4. In the in vivo data, n=4, which does not meet the statistical requirements.

Response
Response: Thank you for your comment. During the revision period, we have supplemented several biocompatible experiments (Figure S30, S31), investigation ( Figure S39), survival experiment (Figure S40), pharmacokinetic experiment ( Figure   S41) with a higher replication number (n = 10) to meet the statistical requirements.
5. The animal survival curve is that n=6-5 does not meet the statistical requirements.
Generally, the number of animals in each group should be 10-20.
Response: Thank you for your comment. The survival experiment has been reconducted with a replication number of 10. Results have been updated in Figure 5c in the revised manuscript.
6. The detection data on autophagy, mitophagy, apoptosis and ferroptosis of cells and animals are insufficient.
Response: Thank you for your comment and suggestion. The present manuscript has been focused on the ferrous traffics and oxidative-associated pathologies during the cardiotoxicity and cardioprotection by MgHCF NCs and DXZ. The authors have elaborated to characterize the possible markers and proteins that are relevant to ferroptosis and apoptosis, including the intracellular free ferrous detection, intracellular oxidative stress and key protein expressions etc. We may have detected the DOXinduced autophagy and mitophagy pathology during the cardiotoxicity investigation using mRNA-seq. However, the main thesis of the present work is on the ferromodulation enabled by MgHCF NCs. We appreciate your understanding.
In short, this study is interesting, but not sufficient to be published in NC. More functional studies and pharmacological studies data should provided to support the key results.
Response: Thank you for your kind comment. Following your suggestions, we have supplemented major functional and pharmacological studies as presented above. We hope that the major revision could well-address your concern.

Reviewer #3 (Remarks to the Author)
In cancer patients, treatment with anthracyclines (i.e., doxorubicin) is very often jeopardized by drug-induced cardiotoxicities mostly caused by excessive cellular oxidative stress. Here, the authors clearly demonstrate that the accumulation of radical species is catalyzed by Fenton-like reactions consecutive to alterations in iron transport and report on the development of a new nanomedicine, called MgHCF NCs, specifically designed to capture iron and hence reduce the iron-associated risk. After Overall, the authors' conclusions (claiming that their new MgHCF Ncs nanomaterial is one of the most promising cardioprotective agents for clinical use) are appropriately supported by the data, well justified and reliable.
Response: Thank you for your comprehensive comments on the validity of our work.
We appreciate the reviewer's time and efforts in reviewing the manuscript.

Significance
To the best of my knowledge, the authors' working hypotheses on the cellular mechanism leading to doxorubicin-induced cardiotoxicity, as well as their conclusions regarding the efficacy of their new nanocatalyst as a promising cardioprotective agent during cancer therapy are correct. I am not aware of any publication supporting contradictory data. Response: Thank you for your comments and suggestions. In the revised manuscript, we have revised "SOD-like activities" to "superoxide radical dismutation activities".
We also rephrased "CAT/catalase like catalytic activities" to "H2O2-decomposition activities" following the reviewer's suggestions. 3. Now, regarding the in vivo data in mice, the biocompatibility was only assessed in a limited number of animals (n= 4/group), which seems too little for conduct appropriate statistical test. In addition, data on the MgHCF NCs' ADME properties are lacking to properly evaluate potential adverse effects. How fast the agent is absorbed after i.p.
injection. What are the plasma halftime and Cmax. To what tissues/organs is the agent distributed ? What is the proportion of the injected compound reaching the target organ (heart) and what is the mechanism of uptake by cardiac cells. For a better risk assessment of the exposure to MgHCF NCs, it is also mandatory to know the expected efficacy dose. This could help in the determination of a margin of safety. Finally, the rationale behind the selection of doses (both for MgHCF NCs and dexrazoxane) used in the in vivo experiments should clearly be stated.
Response: Thank you for your suggestions. We have supplemented a new set of in vivo biocompatibility evaluation (n = 10), in which a body weight profile of mice was recorded. At the end of the evaluation, major blood biochemical indexes and histology examinations were investigated. These supplemented data are presented as Figure S30 in the revised Supporting Information. Relevant discussion has been supplemented in Page 19 in the revised Manuscript.
To evaluate the ADME properties of MgHCF NCs, we have supplemented detailed experiments regarding the pharmacokinetic properties of MgHCF NCs (halfterminal time experiment, tissue biodistribution experiment, etc.) ( Figure S41). We found that upon MgHCF NCs administration, MgHCF NCs were mainly distributed into liver, spleen and lung in 2 h post-injection. In 12 h and 24 h post-injection, the overall distribution amounts of Mg 2+ into these major organs are gradually decreased.
Specifically, the distributed percentages of Mg 2+ into heart were determined to be 7.16 ± 0.39 ID %/g, 5.39 ± 1.77 ID %/g and 1.95 ± 0.27 ID %/g respectively. For tumor accumulation, 2.84 ± 0.84 ID %/g, 1.23 ± 0.45 ID %/g and 1.20 ± 0.52 ID %/g could be determined in 2 h, 12 h and 24 h post-injection, respectively. Relevant discussion has been supplemented in the revised Manuscript (Page 25). where w represents the drug utilization; C0 represents the plasma concentration of Mg 2+ at t = 0; ID represents the injection dose, i.e., 4.8 mg kg -1 ; m represents the weight of mice; V represents the total circulation blood volume of a mice.
The uptake mechanism of the nanomaterials by cardiomyocytes can be determined by the surface charge and chemical composition of the nanomaterials (Small, 2010, 6(1): 12-21). Negatively charged MgHCF NCs may non-specifically bind to the cationic sites on the plasma membrane of the cardiomyocytes with subsequent endocytosis. It has also been indicated that spontaneous contraction of the cardiomyocytes could improve the internalization of the negatively charged nanomaterials due to the K + efflux and subsequent increased membrane potential (Physiological reviews, 2005, 85(4): 1205-1253).
In the median lethal dose investigation, we used concentrated MgHCF NCs for dose biosafety evaluation. Mice received intraperitoneal administration of MgHCF NCs (single dose) at an extremely high dose of 30 mg kg -1 show an overall survival rate of 80 %, while those administrated with lower doses of 15, 10 and 7.5 mg kg -1 all survived for two-weeks. It could be calculated that the median lethal dose of MgHCF NCs for mice is about 52.5 mg kg -1 (Figure S40, discussion on Page 24 in the revised Manuscript). Higher doses of MgHCF NCs are not applicable due to the solubility limit and stability issue.
To rationalize the dose selection of MgHCF NCs, we have determined the effective in vitro dose of MgHCF NCs to the cells as follows by using a MgHCF NCs solution at 40 μg ml -1 . Based on an averaged mice weight of 20 g, total blood volume = 2 mL, and assuming a drug utilization of 20 %, we have determined an injection dose of 20 mg kg -1 . To perform multiple dosing, we have reduced the dose to 5 mg kg -1 and the injection solution was finally calibrated to a magnesium concentration of 4.8 mg kg -1 . The following paper (PNAS, 2019, 116 (7): 2672-2680) has been referenced for the dose selection of DXZ.
4. In the safety assessment of MgHCF NCs in vivo (mice), I regret that the authors did not investigate the potential effect of the magnesium released from nanomedicine during iron uptake. Indeed, a magnesium overload is known to cause cardiovascular effects, including cardiac cytotoxicity and hypotension.
Response: Thank you for your comments and suggestions. After careful literature survey, we have found that it is of great difficulties to in vivo monitor the magnesium release. We also agree with the reviewer's opinion that magnesium overload is potentially harmful to the cardiac functionalities. During MgHCF cardioprotection, magnesium ions have been released from the nanomedicine during iron uptake. Such an ion exchange occurs concurrently and equimolarly. Under the injection dose of MgHCF NCs of 4.8 mg kg -1 d -1 , the highest Mg 2+ flux could be obtained when all of the magnesium ions were released into the cardiomyocytes (i.e., 4.8 mg kg -1 ). Therefore, we employed multiple doses of MgHCF NCs (4.8 mg kg -1 ) or MgCl2 (10 mg kg -1 ) injections to assess the biosafety through echocardiography. In the present investigation, both MgHCF NCs and Mg 2+ exhibit good cardiac biocompatibility during 7 days evaluation timeframe. According to the echocardiographic and electrocardiogram inspections, direct intraperitoneal administration of free Mg 2+ at a dose not higher than negligible under current injection doses of MgHCF NCs ( Figure S39 in the revised

Supporting Information, Page 23 in the revised Manuscript).
In most of the in vitro and in vivo assays (except maybe for the biocompatibility) presented in the study, the number of replicates and statistical tests are appropriate.
Response: Thank you for your comment and suggestions. The biocompatibility experiment has been reconducted with a replication number of 10 ( Figure S30) as presented and described above. We hope that the revision could satisfy your consideration.

Analytical approach
As already mentioned, the analytical methods used in the physico-chemical characterization of the nanomedicine are outside the scope of my expertise. Regarding other analytical approaches, I only have one specific comment related to the assessment of cardioprotection using echocardiography. I do believe that those registered cardiac parameters are not sufficient for a comprehensive risk assessment. In particular, due to the release of Mg++ ions in the cardiac cells during iron uptake by the nanocatalyst, I would recommend to add ECG-like recordings.
Response: Thank you for your comment and suggestions. In the supplemented cardioprotection experiment, we have combined the echocardiography and electrocardiogram inspections to support the analyses ( Figure S39 in the revised Supporting Information, Page 23 in the revised Manuscript).

Suggested improvements
As already mentioned, additional information should be provided regarding : 1. Rationale for the selection of the doses (MgHCF NCs and dexrazoxane) used for the in vivo experiments Response: Thank you for your comments. To rationalize the dose selection of MgHCF NCs, we have determined the effective in vitro dose of MgHCF NCs to the cells as follows by using a MgHCF NCs solution at 40 μg ml -1 . Based on an averaged mice weight of 20 g, total blood volume = 2 mL, and assuming a drug utilization of 20 %, we have determined an injection dose of 20 mg kg -1 . To perform multiple dosing, we have reduced the dose to 5 mg kg -1 and the injection solution was finally calibrated to a magnesium concentration of 4.8 mg kg -1 . The following paper (PNAS, 2019, 116 (7): 2672-2680) has been referenced for the dose selection of DXZ. These experiments could be employed to determine the potential pharmacokinetics of magnesium ions from nanocatalyst in blood compartment and other tissues. Detail data and descriptions have been supplemented in the revised Manuscript (Page 25) and
5. Discuss the potential risk of Mg++ release in cardiomyocytes during iron uploading in the nanocarrier Response: Thank you for your comment and suggestions. We agree with the reviewer's opinion that magnesium overload is potentially harmful to the cardiac functionalities.
During MgHCF cardioprotection, magnesium ions were released from the nanomedicine for iron uptake. Such an ion exchange occurs equimolarly. Under the injection dose of MgHCF NCs of 4.8 mg kg -1 d -1 , the highest Mg 2+ flux could be obtained when all of the magnesium ions were released into the cardiomyocytes (i.e., 4.8 mg kg -1 ). Therefore, we employed multiple doses of MgHCF NCs (4.8 mg kg -1 ) or MgCl2 (10 mg kg -1 ) injection to assess the biosafety through echocardiography. In the present investigation, both MgHCF NCs and Mg 2+ exhibit good cardiac biocompatibility during 7 days evaluation timeframe. According to the echocardiographic and electrocardiogram inspections, direct intraperitoneal of free Mg 2+ at doses not higher than 10 mg kg -1 should be safe, revealing that cardiac toxicity or other abnormalities is negligible under current injection doses of MgHCF NCs